Blood coagulation is a complex process involving many activating and inactivating coagulation factors. Anticoagulant proteins are known to be important for regulation of the coagulation process and anticoagulants are thus important in the treatment of a variety of diseases, e.g. thrombosis, myocardial infarction, disseminated intravascular coagulation etc.
Thus heparin is used clinically to increase the activity of antithrombin III and heparin cofactor II. Antithrombin III is used for the inhibition of factor Xa and thrombin. Hirudin is used for the inhibition of thrombin and protein C may be used for the inhibition of factor V and factor VIII. Anticoagulant proteins may also be used in the treatment of cancer.
Coagulation can be initiated through the extrinsic pathway by the exposure of tissue factor (TF) to the circulating blood (Y. Nemerson, Blood 71 (1988) 1-8). Tissue factor is a protein cofactor for factor VII/VIIa and binding of tissue factor enhances the enzymatic activity of factor VIIa (FVIIa) towards its substrates factor IX and factor X.
Recently a new anticoagulant protein, the tissue factor pathway inhibitor (TFPI) has been isolated (G. J. Broze et al., Proc. Natl. Acad. Sci. 84 (1987) 1886-1890).
On a molar basis TFPI has been shown to be a potent inhibitor of TF/FVIIa-induced coagulation (R. A. Gramzinski et al., Blood 73 (1989) 983-989). TFPI binds and inhibits factor Xa (FXa) and the complex between TFPI and FXa inhibits TF/FVIIa (Rapaport, Blood 73 (1989) 359-365). TFPI is especially interesting as an anticoagulant/antimetastatic agent as many tumor cells express TF activity (T. Sakai et al., J. Biol. Chem. 264 (1989), 9980-9988) and because TFPI shows anti-Xa activity like antistatin which has antimetastatic properties.
TFPI has been recovered by Broze et al. (supra) from HepG2 hepatoma cells (Broze EP A 300988) and the gene for the protein has been cloned (Broze EP A 318451). A schematic diagram over the secondary structure of TFPI is shown in U.S. Pat. No. 5,312,736 (WO 91/01253). The amino acid sequence of TFPI with its natural 28 amino acid signal peptide (Sequence ID Number 1 and 2) is shown in FIG. 1 where the N-terminal amino acid Asp is given the number 1. The protein consists of 276 amino acid residues and has in addition to three inhibitor domains of the Kunitz type, three potential glycosylation sites at position Asn117, Asn167 and Asn228. The molecular weight shows that some of these sites are glycosylated. Furthermore, it has been shown that the second Kunitz domain binds FXa while the first Kunitz domain binds FVIIa/TF (T. J. Girard et al., Nature 338 (1989) 518-520). TFPI has also been isolated from HeLa cells (PCT/DK90/00016) and it was shown that HeLa TFPI binds heparin.
In U.S. Pat. No. 5,312,736 certain TFPI analogues are described retaining the TFPI activity as well as anti Xa activity although parts of the molecule have been deleted. Furthermore, these analogues show a much lower affinity for heparin than full length TFPI, making them more useful as therapeutic agents than the native molecule. The TFPI analogues will furthermore have a longer half life as compared with native TFPI which will further reduce the amount of active ingredients for the medical treatment.
These TFPI analogues are thus characterized in having TFPI activity but with no or low heparin binding capacity under physiological conditions (pH, ionic strength).
In the present context the term "low heparin binding capacity" is intended to mean a binding capacity of about 50%, more preferably of about 25% and most preferably less than about 10% of that of native TFPI at physiological pH and ionic strength.
It was thus shown that the heparin binding capacity is lost when the sequence from amino acid residue number 162 to amino acid residue number 276 is deleted from the TFPI molecule. It was therefore concluded that the heparin binding domain is situated in this part of the TFPI molecule and it was assumed that the heparin binding domain comprises at least a region from Arg246 to Lys265 near the C-terminal end of the TFPI molecule being rich in positively charged amino acid residues.
The present invention is based on the surprising fact that active TFPI analogues lacking C-terminal parts of the molecule are expressed in good yields in yeast. This is the more surprising because attempts to express native TFPI in yeast gave only neglible amounts.